Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans

Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60°C and pH 7.5 and at 50°C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49h incubated at 50°C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC

Purification and characterization of xylanases from the fungus Chrysoporthe cubensis for production of xylooligosaccharides and fermentable sugars

Xylanases from the pathogen fungus Chrysoporthe cubensis were produced under solid state fermentation (SSF) using wheat bran as carbon source. The enzymatic extracts were submitted to ion exchange (Q Sepharose) and gel filtration chromatography methods (Sephadex S-200) for purification. The xylanases were divided into three groups: P1 showed better performance at 60 °C and pH 4.0, P2 at 55 °C and pH 3.0, and P3 at 80 °C and pH 3.0. Oat spelt xylan was the best substrate hydrolyzed by P1 and P3, while beechwood xylan was better degraded by P2. Carboxymethyl cellulose (CMC) and p-nitrophenyl-β-d-xylopyranoside (p-NPβXyl) were not hydrolyzed by any of the xylanases. The K M ’ or K M values, using oat spelt xylan as substrate, were 2.65 mg/mL for P1, 1.81 mg/mL for P2, and 1.18 mg/mL for P3. Xylobiose and xylotriose were the main xylooligosaccharides of oat spelt xylan degradation, indicating that the xylanases act as endo-β-1,4-xylanases. Xylanases also proved to be efficient for hydrolysis of sugarcane bagasse when used as supplement of a commercial cocktail due to the increase of the reducing sugar release

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32g/L) and xylose (9.00g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84U/mg of protein), β-glucosidase (29.48U/mg of protein), β-xylosidase (2.95U/mg of protein), pectinase (127.46U/mg of protein) and laccase (2.49U/mg of protein)

The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails : A comparative study

Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32g/L) and xylose (9.00g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84U/mg of protein), β-glucosidase (29.48U/mg of protein), β-xylosidase (2.95U/mg of protein), pectinase (127.46U/mg of protein) and laccase (2.49U/mg of protein)

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant-degrading enzymes can be produced by fungi on abundantly available low-cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two "second generation" substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi-)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non-washed SCB. The sensitivity to non-washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification

Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free β-glucosidase

An intracellular β-glucosidase from Debaryomyces hansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15 kDa. Yeast cells containing the intracellular β-glucosidase were immobilised in calcium alginate. The free β-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45 °C and 5.5 and 50 °C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90 days at 4 °C and after 15 days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50 °C. Soy molasses treatment with the free enzyme and the immobilised cells containing β-glucosidase, for 2 h at 40 °C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses

Characteristics of free endoglucanase and glycosidases multienzyme complex from Fusarium verticillioides

A novel multienzyme complex, E1 C , and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1 C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1 C and E2 were 21.3 and 27.5 kJ/mol, respectively. The K M for E1 C was 10.25 g/L while for E2 was 6.58 g/L. Both E1 C and E2 were activated by Mn 2+ and CoCl 2 while they were inhibited by SDS, CuSO 4 , FeCl 3 , AgNO 4 , ZnSO 4 and HgCl 2 . E1 C and E2 presented endo-b-1,3–1,4-glucanase activity. E1 C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1 C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins